Download The Isolation and Characterization of Gallium

[CANCER
RESEARCH
33, 2063-2067, September 1973]
The Isolation and Characterization of Gallium-binding Granules from
Soft Tissue Tumors'
David H. Brown, Donald C. Swartzendruber,
and Raymond 1. Hayes
James E. Carlton, Billy 1. Byrd,
Medical Division, Oak Ridge Associated Universities,2 Oak Ridge, Tennessee 37830
@
SUMMARY
Both rate-zonal and isopycnic-zonal centrifugation exper
iments indicate that 67Ga in rat tumors (RFT tumor and
Morris 7777, 5l23C, and 7794A hepatomas) and in a mouse
lymphosarcoma
(P- 1798) is associated with lysosomal gran
ules. The particles were identified enzymatically by their
acid phosphatase
and N-acetyl-@3-D-glucosaminidase
con
tent. Autoradiography
of selected gallium-binding granule
fractions showed silver grains concentrated over electron
dense single-membrane
organelles.
INTRODUCTION
Many different types of tumors in animals and man have
an affinity for gallium. It is possible to detect such tumors
by scintiscanning techniques using the radionuclide 67Ga (5,
8, l I). Elucidation of the mechanism of 67Ga uptake by
tumor tissue should reveal those factors that control its
localization and possibly lead to methods for enhancement
of its affinity for cancers.
Intracellular 67Ga present in normal and neoplastic tissue
1 to 2 days after i.v. administration
has been shown by
with RFT tumors (9); and female BALB/c x DBA/2
mice with P-1798 lymphosarcomas.
Animals were main
tamed on the same diet and fed ad libitum.
Twenty-four to 48 hr after the administration of 67Ga (‘—j
I
mCi/kg), the animals were lightly anesthetized with ether
and the tumor tissues were excised. The tissues were rinsed
with 50 ml of 0.25 M sucrose and then homogenized in
cold (5°)0.25 M sucrose (10% w/v) in a Potter-Elvehjem
type homogenizer operating at 1000 rpm (5 strokes), a
procedure considered acceptable for release of subcellular
organelles with minimal loss of intact particles resulting
from the tissue-rupturing
technique (4). Nuclei and cell
debris were removed from the homogenate by centrifuga
tion in a Sorvall GLC-l centrifuge for 2500 g-min. Sub
cellular particles from the homogenates
(60 to 100 ml)
were then isolated by rate-zonal centrifugation in a B-XXIX
zonal rotor (loaned by Dr. N. G. Anderson, MAN Pro
gram, Oak Ridge National Laboratory) in a I-liter gradient
of 20 to 30% w/w sucrose using our sequential product
recovery technique (2). The rotor effluents were monitored
at 260 nm. All operations were carried out at 5°.
A modificationof the zonal centrifugationprocedureof
Leighton et al. (12) was also used to isolate lysosomes. Male
Buffalo rats bearing Morris 5l23C hepatomas were given
EM-ARG3 to localizein lysosome-likebodiesin a varietyof injections i.v. of the nonionic detergent Triton WR-l339
cell types (14). The rate-zonal centrifugation and enzymatic
(Rohm and Haas Co., Philadelphia, Pa.) 2 days before i.v.
studies presented here provide further evidence of the injection of °7Ga.Twenty-four hr after giving 67Ga we killed
lysosomal nature of the GBG previously identified by the animals and removed and homogenized the tumors as
EM-ARG.
described
above. (A control experiment
in which no
WR-1339 was administered was also performed.) The brei
was then centrifuged for 10,000 g-min to remove the cell
debris
and nuclei. The supernatant (100 ml) was layered
MATERIALS
AND METHODS
over a 500-ml sucrose gradient (20 to 25%) in an Oak Ridge
type B-XXIX zonal rotor. Outboard of the gradient were 3
67Ga (Oak Ridge National
Laboratory,
Oak Ridge,
Tenn.) was prepared as the neutral citrate and injected i.v. in steps consisting of 300 ml of 35%, 200 ml of 45%, and 227
the tail vein ( 1 mg citrate per kg). Detection of the 67Ga ml of 55% sucrose. The centrifuge was operated at 25,000
rpm for a total of 4.3 x 106 g-min (3,750 x l0@ w2t). All
present in gradient fractions was done in a well-type
operations were carried out at 5°.
scintillation counter.
Fractions from the centrifugations were assayed for acid
The following animals and transplanted
tumors were
phosphatase
(EC 3. 1.3.2) and N-acetyl-@3-D-glucosamini
used: male Buffalo rats implanted with Morris 7794A,
dase
(EC
3.2.1.30)
by the automated method described by
5l23C, and 7777 hepatomas; male Fischer rats implanted
Beck ( 1), protein was measured by an automated modified
Lowry procedure (6), and sucrose concentratlons were deter
CA-l 1858 from the National Cancer Institute.
mined refractometrically.
A method described by Hinton et
al. (10) was used to correct for sucrose error in the enzyme
3The abbreviations used are: EM-ARG, electron microscopic autoradi
and protein determinations.
Electron microscopy and EM
ography; GBG, gallium-binding granules.
ARG were performed on the GBG fractions; fixation,
Received December 27, 1972; accepted May 15, 1973.
1 This
investigation
2 Under
contract
SEPTEMBER
was
with
the
supported
U.
S.
by
Atomic
USPHS
Energy
Research
Grant
Commission.
1973
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.
2063
Brown, Swartzendruber,
Car/ton, Byrd, and Hayes
staining, and autoradiography
previously (14).
RESULTS
were performed
as described
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4
AND CONCLUSIONS
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The zonal profiles of homogenates (cleared of nuclei and
cell debris) of 3 different tumors, a poorly differentiated rat
tumor (RFT), a mouse lymphosarcoma
(P-1798), and a
Morris rat hepatoma (7777), are shown in Chart I. The
gradient profiles of the 67Ga activity shown in Chart 1
indicate that the GBG were primarily located in the
6th-stage particle zone. It is apparent that the GBG from
each of the tumors sedimented at similar rates in the
gradients and therefore had similar sedimentation
coeffi
cients. 67Ga recoveries from the rotor varied from 85 to
95%.
To determine whether any of the radioactivity remaining
in the starting zone would sediment after longer centrifuga
tion, we removed the 6th-stage particle zone (as in the other
centrifugal integrals) and replaced it with an equal volume
of 45% sucrose; the rotor was then reaccelerated to 25,000
rpm and held there 17 hr for a centrifugal integral of 42,300
x
l0@
w@t.
It
is
evident
from
the
results
of
the
RFT
experiment
shown in Chart 1 that only a very small
percentage of the 67Ga activity banded on the 45% sucrose
cushion after this additional centrifugal integral.
Enzymatic evidence for the lysosomal nature of the
6th-stage GBG is shown in Chart 2 for a Morris 7794A
hepatoma. The greatest relative specific activity (percentage
of total enzyme activity divided by percentage of total
protein) was found in the 6th-stage particle fractions.
Lysosomes were, however, distributed in all the particle
zones. Recoveries of protein, acid phosphatase, N-acetyl-f3-
6.0
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8.0
a.
2.'
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@0
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z
6.0 0
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a.
FRACTION
NUMBER
Chart I . Distribution of “7Ga
in subcellular fractionations of 3 different
transplanted animal tumors. The particles were separated by a discontinu
ous rate-zonal centrifugation procedure in an edge-unloading zonal rotor
(B-XXIX).
The direction of sedimentation is from right to left. The
absorbance profile (260 nm) of the rotor effluent is shown as a dashed line.
The histograms (solid lines) show the distribution of the ‘7Ga.
The peaks at
the right of the charts represent those components that remain in the
starting zone.
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Ui
100
50
0
20
30
40
50
FRACTIONNUMBER(4OmI each)
Chart 2. Rate-zonal particle profile of a Morris 7794A rat hepatoma
showing the relative specific activities (percentage of total enzyme in
fraction divided by percentage oftotal protein in fraction) ofthe lysosomal
enzymes, N-acetyI-@-o-glucosaminidase and acid phosphatase.
D-glucosaminidase,
and 67Ga were, respectively, 96, 97, 90,
and 104%.
We believe the radioactivity normally remaining in the
starting zone after the centrifugal integral of 800 x l0@w@t
(Charts 1 and 2) to be mostly non-particle-bound
67Ga,
probably arising from broken lysosomes and plasma 67Ga
not removed in the rinse procedure before homogenizing.
We obtainedfurtherevidencefor the lysosomalnatureof
the GBG by isolating “light―
lysosomes by isopycnic-zonal
centrifugation
by the method of Leighton et a/. (12). The
procedure intrinsically depends upon modifying the density
of lysosomes in experimental animals. The nonionic deter
gent Triton WR-1339 is injected 2 to 3 days prior to
administration
of 67Ga. This material is taken up by
lysosomes causing them to become less dense than the
mitochondria with which they “normally―
band. Hence, one
can separate the 2 organelles from each other by isopycnic
centrifugation.
The density of such altered lysosomes is
reported to be 1. I3 g/ml ( 12). The results of a modified
version ofthis procedure are shown in Chart 3. Only a small
percentage of the total protein (4%) occurred in the “light―
lysosomal zone (p = I . 135) and the acid phosphatase
activity of the particles in this zone was quite high (22%), as
was the 67Ga activity (27%) compared
to the control
experiment (no administered WR-1339) where only 12% of
the acid phosphatase activity and 8% of the 67Ga activity
were found. A comparison
of the relative specific acid
phosphatase activities of the light lysosomes regions of the
gradients in the 2 experiments [65 (+WR-l-339),
17 (—WR
1339)] indicated that the tumor tissue lysosomes did take up
Triton WR-1339. The percentage of 67Ga was also greater
in the zone where the light lysosomes banded (see above).
It is also evident, however, that the more dense particles
contained a large amount of acid phosphatase (27%) and
67Ga activity (21%). Leighton et a/. (12) found normal as
well as light lysosomes in WR-1339-treated
animals, which
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Characterization
60
mitochondria;
(b) vesicles of rough-surfaced
endoplasmic
reticulum; and (c) single-membrane-limited,
electron-dense
structures about 0.25 to 0.5 zm in diameter. More silver
grains were found over the regions containing the most
lysosomes and fewer silver grains were observed over
regions of the pellet in which mitochondria and vesicles of
rough-surfaced endoplasmic retculum predominated.
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DISCUSSION
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FRACTION
NO. (40
30
ml)
Chart 3. The association of “7Ga
with “light―
lysosomes isolated by
isopycnic-zonal centrifugation from a Morris 5123C rat hepatoma. The
upper trace in the chart shows the concentration of sucrose in the rotor as
determined from refractive index measurements of the collected samples.
Sedimentation of the particles was from right to left. The relative specific
activity of acid phosphatase (90% recovery) is shown by the dotted
histogram at the bottom of the chart. The total ‘7Ga
radioactivity (82%
recovery) of each fraction is shown as a solid-line histogram. The recovery
of protein was 104%.
could account for the acid phosphatase and 67Ga activity
seen in the heavier particles. The large amounts of 67Ga
(49%) and acid phosphatase (46%) activity in the soluble
“zone―
could be accounted for by the rupturing of some of
the light lysosomes as a result of increased fragility when
they take up the detergent. The control experiment showed
only 22% acid phosphatase activity and 10% 67Ga in this
zone. Recoveries in this study are indicated in the legend to
Chart 3.
Electron microscopic autoradiograms
made from thin
sections of isolated 6th-stage GBG showed silver grains
predominantly
localized over small electron-dense
or
ganelles limited by single membranes (Figs. 1 and 2). The
granules associated with the silver grains were morphologi
cally similar to those identified previously as lysosomes in
67Ga autoradiograms
of murine tissues (14). Three distinct
regions in the pellets obtained from the 6th-stage fractions
were distinguished by ultrastructural
study. These were: (a)
SEPTEMBER
Swartzendruber
et a/. (14), using EM-ARG, have shown
that intracellular
67Ga present in normal and neoplastic
tissue 1 to 2 days after i.v. administration
is localized in
lysosome-like bodies in a variety of cell types. The subcellu
lar fractionation and enzymatic studies reported here for 4
different transplanted tumors provide further evidence for
the lysosomal nature of the organelles previously identified
by EM-ARG.
Contrary to the results that we report here, those of
Deckner et al. (3) with Ehrlich ascites cells and Orii (13)
with Yoshida sarcoma have indicated that little or no 67Ga
is associated with lysosomes. Orii also reported that no
accumulation of 67Ga was observed in liver lysosomes, also
contrary to our previous findings (2) as well as those of
Haubold and Aulbert (7). Orii's results and those of
Deckner et a/. may, therefore, be due to the disruption of
lysosomes in some phase of the fractionation
procedures
that they used.
REFERENCES
1. Beck, C. I. Lysosomal Enzymes; Chromatography and Automated
Methods. Dissertation, University of California, Davis, Calif., 1967.
2. Brown, D. H., Carlton, E., Byrd, B., Harrell, B., and Hayes, R. L. A
Rate-Zonal Centrifugation Procedure for Screening Particle Popula
tions by Sequential Product Recovery Utilizing Edge-Unloading
Zonal Rotors. Arch. Biochem. Biophys., 155: 9-18, 1973.
3. Deckner, K., Becker, G., Langowski, U., Schwering, H., Hornung, G.,
and Schmidt, C. G. The Subcellular Binding of 67-Gallium in Ascites
Tumor Cells. Z. Krebsforsch., 76: 293-298, 1971.
4. Dc Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and
Applemans, F. Tissue Fractionation Studies. VI. Intracellular Distri
bution Patterns of Enzymes in Rat-Liver Tissue. Biochem. J., 60:
604-617,1955.
5. Edwards, C. L., and Hayes, R. L. Scanning Malignant Neoplasms
with Gallium-67. J. Am. Med. Assoc., 212: 1182—1190,1970.
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1966, to June 30, 1967, U. S. Atomic Energy Commission Report
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Laboratory, 1967.
7. Haubold, U., and Aulbert, E. “7Ga
as a Tumor Scanning Agent
Clinical and Physiological Aspects. In: Medical Radioisotope Scintig
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8. Hayes, R. L., and Edwards, C. L. New Applications of Tumor-Local
izing Radiopharmaceuticals. In: Medical Radioisotope Scintigraphy.
Vienna: International Atomic Energy Agency, in press.
9. Hayes, R. L., Nelson, B., Swartzendruber, D. C., Carlton, J. E., and
Byrd, B. L. Gallium-67 Localization in Rat and Mouse Tumors.
Science,167:289—290,
1970.
1973
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2065
Brown, Swartzendruber,
Car/ton, Byrd, and Hayes
10. Hinton, R. H., Burge, M. L. E., and Hartman, G. C. Sucrose
Interference in the Assay of Enzymes and Protein. Anal. Biochem., 29:
248—256,1969.
11. Langhammer, H., Glaubitt, G., Grebe, S. F., Hampe, J. F., Haubold,
U., Hör,G., Kaul, A., Koeppe, P., Koppenhagen, J., Roedler, H. D.,
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for Tumor Scanning. J. NucI. Med.,
13: 25-30, 1972.
12. Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey,J., Fowler,
2066
S., and Dc Duve, C. The Large-Scale Separation of Peroxisomes,
Mitochondria, and Lysosomes from the Livers of Rats Injected with
Triton WR-1339. J. Cell Biol., 37: 482-513, 1968.
13. Orii, H. Tumor Scanning with Gallium (“7Ga)
and Its Mechanism
Studied in Rats. Strahlentherapie, 144: 192-200, 1972.
14. Swartzendruber, D. C., Nelson, B., and Hayes, R. L. Gallium-67
Localization in Lysosomal-Like Granules of Leukemic and Non
leukemic Murine Tissues. J. Natl. Cancer Inst., 46: 941-952, 1971.
CANCER
RESEARCH
VOL.
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33
‘I
Fig. I. An electron microscopic autoradiogram of rat tumor(RFT) 6th-stage fraction containing the majority of ‘7(Ia
activit@. This area ofthe block
contained more lysosomes and more silver grains than adjacent regions. x 3 1.000.
Fig. 2@An electron microscopic autoradiogram of 6th-stage fraction from mouse Iymphosarcoma (P-l79@) shos@ingmans silver grains localized in a
region containing mans lysosomes. A few mitochondria and vesicles of rough-surfaced endoplasmic reticulum are also present. x 29.000.
SEPTEMBER
1973
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.
2067
The Isolation and Characterization of Gallium-binding Granules
from Soft Tissue Tumors
David H. Brown, Donald C. Swartzendruber, James E. Carlton, et al.
Cancer Res 1973;33:2063-2067.
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